Taxonomic Applicability

Sex Applicability

Life Stage Applicability

Key Event Relationship Description

Cyp2E1 activation has two major outcomes: (1) the production of reactive, electrophilic metabolites, and (2) a significant increase in the half-life of the Cyp2E1 enzyme (Gonzalez 2007, Song, et al. 1989). The former is important because metabolites can go on to produce cellular damage by reacting with cellular nucleophiles. The latter is important because the Cyp2E1 catalytic cycle is prone to uncoupling (i.e., instead of incorporating an oxygen atom in to the substrate, the catalytic cycle is interrupted because a superoxide radical is released), which results in the release of reactive oxygen species (ROS) and an increase in cellular oxidative stress (Lieber 1999).

Evidence Supporting this KER

Biological Plausibility

It is well known that uncoupling of Cyp2E1 catalytic cycle results in the release of harmful reactive oxygen species in the cell (Lieber 1999).

Oxidative stress is produced during chronic activation (and uncoupling) of the Cyp2E1 catalytic cycle. The cytochrome P-450 catalytic cycle is known to undergo uncoupling leading to the production of ROS (Gorsky, et al. 1984, Loida and Sligar 1993, Meunier, et al. 2004). If this uncoupling occurs, a molecule of superoxide radical is released, which has the effect of interrupting the P450 catalytic cycle and releasing harmful ROS into the cell. Typically superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD), which is further reduced into the hydroxyl radical (OH•), and then to water. Other relevant cellular antioxidants include glutathione, thioredoxin, and peroxiredoxins. However, it is also possible for these ROS to scavenge electrons from cellular macromolecules (proteins, lipids, nucleic acids). Because Cyp2E1 is membrane-bound, ROS most commonly react with lipids and initiate lipid peroxidation. Further, Cyp2E1 can undergo NADPH-dependent ‘futile cycling’, which produces ROS and contributes to the occurrence of lipid peroxidation (Ekstrom and Ingelman-Sundberg 1989). Persistent oxidative stress also creates opportunities for oxidative damage to DNA, which is important because DNA damage is required for the initiation phase of carcinogenesis. The cellular sources and effects of ROS, as well as the corresponding enzymes and antioxidants are reviewed in:. (Nakazawa, et al. 1996).

Empirical Evidence

Empirical data collected from different experiments strongly supports that oxidative stress and cytotoxicity are downstream of Cyp2E1 activation. Evidence of both temporal and dose-response concordance are available for a variety of chemical exposures.

Cyp2E1 protein levels increase when its substrate is present in a tissue. Therefore, prolonged exposure to substrate leads to prolonged activation of Cyp2E1, which is related to a substantial increase in cellular oxidative stress. For example, treatment with acetone or ethanol in male Sprague-Dawley rats results in an increase in Cyp2E1 protein levels in the liver. Increasing Cyp2E1 levels are linearly correlated to concomitant increases in NADPH oxidase, superoxide radical, hydrogen peroxide, and lipid peroxidation (TBARS). Both hydrogen peroxide production and lipid peroxidation are blocked in rat microsomes following inhibition of Cyp2E1 with anti-Cyp2E1 IgG (Ekstrom and Ingelman-Sundberg 1989). Ethanol treatment leads to correlated increases in both Cyp2E1 protein and lipid peroxidation in male Wister rats, C57BL/129SV mice, and superoxide dismutase (Sod) knockout mice (Kessova, et al. 2003, Nanji, et al. 1994). Wild type and humanized Cype2E1 knock-in mice have dose-dependent increases in Cyp2E1 protein and activity levels when exposed to ethanol, whereas Cyp2E1 knock-out mice do not. Further, the humanized mice show the largest increases in necrosis, inflammation, AST, ALT and TBARS, and the largest decrease in GSH levels of all three groups (Lu, et al. 2010). Exposure of male Sprague-Dawley rats to 95% oxygen results in a time-dependent increase in Cyp2E1 protein levels, superoxide radical production, and lipid peroxidation (TBARS) (various time-points over 78 hours). Lipid peroxidation is further increased following treatment of rat microsomes with carbon tetrachloride, or co-treatment of rats with oxygen, acetone and/or carbon tetrachloride (Tindberg and Ingelman-Sundberg 1989), established Cyp2E1 substrates. Cyp2E1 expressing HepG2 cells (called E47 cells) have higher baseline expression of anti-oxidant molecules thioredoxin and glutathione compared to non-Cyp2E1 expressing HepG2 cells (called C34 cells); they also have higher levels of ROS and lipid peroxidation (Yang, et al. 2011). Studies of ethanol- and Cyp2E1-dependent oxidative injury in HepG2 E47 and C34 cells, and in the liver, have been reviewed previously (Caro and Cederbaum 2004, Lu and Cederbaum 2008).